1. Field of the Invention
The present invention relates to confocal microscopes.
2. Description of the Prior Art
Conventional confocal microscopes use a point source that is imaged onto the object that is to be imaged. Then, another imaging system images that part of the object through a pinhole onto a detector. By doing this, the resolution of the final image is increased because the impulse responses (or point spread functions) of each part of the system (the first imaging subsystem that images the source onto the object and the second imaging subsystem that images the object region onto the detector) are multiplied. This gives a narrower overall point spread response, and hence, better resolution.
The primary use of a confocal microscope, however, is to provide an image of a plane of the object without interference from other planes. That is, the confocal microscope provides an image of a slice of the object. This is accomplished because when the image of the point source is re-imaged by the second imaging subsystem onto the pinhole in front of the detector, only the light that comes from the plane being examined is focused through the pinhole. Any light from other planes spreads out rapidly and a negligible portion gets through the pinhole.
In order to image the whole plane being examined, both the point source and the pinhole in front of the detector must be scanned over a two dimensional array of positions corresponding to the slice. Alternatively, the object may be moved in two dimensions transverse to the optical axis.
Then, to examine another slice of the object, the point source is focused onto the new slice, and the scanning process is repeated. Repeating this process gives images of several slices in a volume. Alternately, the object can be moved along the optical axis.
The process of examining even a single slice of the object is very time consuming (much less many slices). Of course it works poorly with live, moving objects, because they move during the scanning time and distort the image.
Other confocal microscopes have speeded up the scanning process to some extent by using an array of micro mirrors to illuminate isolated points or by using spinning wheels with several holes in them, so that several point images can be detected at once. These point images must be far apart so that light directed at one detector pinhole does not go through a different detector pinhole when light from other planes is considered. This causes cross talk. A similar approach uses lenslett arrays to provide multiple point images. Again, the point images must be far apart. Thus, the scanning process is still very slow.
A third approach is to use structured illumination with fine detail that blurs quickly with misfocus. This approach requires multiple exposures to remove the structured illumination from the images.
A need remains in the art for faster confocal microscopes.
An object of the present invention is to provide an improved, faster, confocal microscope. The present invention accomplishes this goal by utilizing an array of light sources imaged onto the object, and an array of small detectors to detect the light from each source. The light could be visible, infrared, or ultraviolet. Cross talk between the beams of light is prevented by temporally modulating the sources at different frequencies.
Light from one source is thus temporally modulated at a first frequency, for example in the megahertz region. A reference signal at the same frequency plus a frequency offset is also sent to the detector assigned to that source. The detected signal and the reference signal are then beat together, and heterodyne detection and a band pass filter are used to detect only the light from the assigned source. The light from the source will beat with the reference signal and produce a signal at the offset frequency. This signal is passed by the bandpass filter. Light from other sources, which is modulated at still different frequencies, will beat with the reference signal to produce different beat signals, which can be filtered out using the band pass filter.
The detector array must be a type that can provide heterodyning, such as photodiode arrays, photo multipliers with arrays of sensor areas, and CMOS detector arrays. The array of sources must comprise near point sources, for example an array of surface emitting lasers. As an alternative, the source array may comprise liquid crystal modulators illuminated with the desired illumination wavelength. The image of the source array that appears on the object can be demagnified to provide the desired resolution.
To image a different plane of the object, either the object is moved (with respect to the source array), or the lenses refocused, as in a conventional confocal microscope.
The number of distinct temporal modulation frequencies that are required is determined by the region of the detector array over which light from one source overlaps onto detectors other than the one intended for that source. This is, in turn, determined by the thickness of the object being imaged. Sources may be modulated with the same frequency, so long as they are far enough apart to not overlap on the detector array.